Injectable and biodegradable piezoelectric hydrogel for osteoarthritis treatment

Osteoarthritis affects millions of people worldwide but current treatments using analgesics or anti-inflammatory drugs only alleviate symptoms of this disease. Here, we present an injectable, biodegradable piezoelectric hydrogel, made of short electrospun poly-L-lactic acid nanofibers embedded inside a collagen matrix, which can be injected into the joints and self-produce localized electrical cues under ultrasound activation to drive cartilage healing. In vitro, data shows that the piezoelectric hydrogel with ultrasound can enhance cell migration and induce stem cells to secrete TGF-β1, which promotes chondrogenesis. In vivo, the rabbits with osteochondral critical-size defects receiving the ultrasound-activated piezoelectric hydrogel show increased subchondral bone formation, improved hyaline-cartilage structure, and good mechanical properties, close to healthy native cartilage. This piezoelectric hydrogel is not only useful for cartilage healing but also potentially applicable to other tissue regeneration, offering a significant impact on the field of regenerative tissue engineering.

1.The title of the manuscript can be re-considered so the main content can be reflected precisely while the novelty is highlighted.For instance, there is no experimental evidence in this manuscript to support the "biodegradable" and the other "medical applications" (except the cartilage regeneration), despite the injectable piezoelectric hydrogel and this implantation strategy certainly have these potentials.2. Section "Piezoelectric hydrogel for chondrogenesis in vitro study": In this section, three different concentrations of NF-sPLLA in hydrogel, i.e., 1, 5, and 10 mg/ml, were given to explore the optimal concentration for subsequent in vitro and in vivo biomedical experiments.As demonstrated in the manuscript: "At a higher amount of NF-sPLLA, there were more ACAN and SOX9 generated (increasing from 1.5 to 10-fold, from 1 mg/ml to 10 mg/ml).However, for COL2A1 gene, 5 mg/ml of the NF-sPLLA generated more COL2A1 genes compared to other groups.",the 10 mg/mL seems to be more effective compared to 5 mg/mL since the group of 10 mg/mL can induce ACAN and SOX9 genes to the optimal state while only the COL2A1 gene exhibits the optimal state when 5 mg/mL is employed.Nevertheless, as elucidated in the manuscript: "Since collagen II is the most abundant (~ 95%) and important protein in the hyaline cartilage matrix, we selected NF-sPLLA concentration of 5 mg/ml for all following experiments and in vivo studies.", the group of 5 mg/mL was selected for subsequent experiments according to the collagen II.How can this contradiction be explained?Another relevant concern is that I fail to find the data with respect to collagen II in the section discussing the optimal concentration.3. Section "Piezoelectric hydrogel induces cartilage healing in rabbit osteochondral defect model": In the section of animal experiment, three different sensors of polyimide, PLLA, PZT were selected at the beginning to confirm whether the 40 kHz ultrasound could penetrate through tissue and reach the target site.Why don't choose the piezoelectric PLLA and non-piezoelectric PDLLA based hydrogels which have been used to carry out the following animal experiment?The result can also be the direct evidence of that the ultrasound certainly activate the piezoelectric hydrogel to generate electrical signal.When it comes to the data obtained from the ultrasound activated polyimide, PLLA, PZT sensors, as illustrated in Figure S6.b, the difference of output voltage was observed between PLLA and PZT.Nevertheless, it's been reported that PLLA and PZT have a huge disparity on the capability of electromechanical conversion.To be specific, PLLA features a piezoelectric constant of ~10 pC/N (https://doi.org/10.1002/adma.201802084)while the commercial PZT have a piezoelectric constant of 450-650 pC/N (https://doi.org/10.1093/nsr/nwac101).Under the activation of the ultrasound with same intensity and frequency, a more remarkable difference should be obtained on the output voltage of PLLA and PZT.However, the difference of output voltage demonstrated in S6.b is not that large.Can this phenomenon be explained as well? 4. Section "Conclusion/Outlook": At the end of this section, three different advantages of the research were concluded, as illustrated in the manuscript: "we have presented, for the first time, a novel piezoelectric hydrogel which can (1) be injected into the body via a minimally invasive process to preclude 440 implantation surgery, (2) self-generate electrical cues to promote cartilage and other tissue healing under US activation, and (3) eventually, degrade into safe degradation byproducts to avoid invasive removal surgery and any harm to the body."However, the corresponding data supports over the concluded advantage (3) and part of (2) (i.e., "other tissue healing") are absent, hence, these parts can't be involved in the conclusion as the highlights. 5.The whole manuscript should be carefully rechecked to remove format errors and typos.For example, as illustrated in Figure 1.d, the numbers on the left axis are covered.Furthermore, there is an inconsistency between the scale bars on Figure 1.b and Figure 1.g-i.
Reviewer #3: Remarks to the Author: The goal of this study is to evaluate the benefits of an injectable, biodegradable piezoelectric hydrogel for cartilage repair.It is interesting, building up on previous work (ref. 55).However, I have the following points that need critical attention: 1. in vitro: 1a. the authors should have used rabbit BM-MSCs instead of ADSCs as those are the local regenerative cells that will repopulate the defects following gel injection.1b.I could not find the details on the US conditions employed.Also, do they match with those applied in vivo?How can they be standardized so they would match?1c.No data are provided on live/dead cells upon (i) US treatment, (ii) hydrogel application, and (iii) both treatments.
2. in vivo: 2a.I could not find the details on the amount of hydrogel applied.Does it match with the condition used in vitro?How can it be standardized so both would match (especially in terms of cell numbers: cell numbers in vitro to BM-derived regenerative cells in vivo)?2b. Figure 5a: it is hard to understand how the US treatment on itself allows for a better adhesion of the hydrogels to the defects (regardless of piezo/non-piezo or even control); the hydrogel is made of unnatural compounds (PLLA, PDLLA, type-I collagen and not hyaline type-II collagen) so it is again difficult to understand that repair occurs with so much matrix formation just upon US (US-piezo-safO-2 months) versus non-US-piezo-safO-2 months.Non-piezo without US is better over time than with US, please explain.Piezo without US is worse over time, please explain (that would not be good if US do not work or are not well tolerated in patients).2c.So, please consider the following: Also here, no data are provided on live/dead cells upon (i) US treatment, (ii) hydrogel application, and (iii) both treatments (TUNEL assay?Caspase assay?).Please include an evaluation of the expression of key matrix compounds (collagens II, I, and X for hypertrophy).Will the US conditions be applicable in patients, will they work, will they be deleterious?
As seen in Figure 1.e the NF-sPLLA dried hydrogel sensor generates a clear signal with consistent intervals and peak magnitude.Meanwhile, NF-sPDLLA dried hydrogel sensor's waveform has smaller amplitude and irregularity with random peaks under the same applied US intensity.Figure 1.f indicates that the output voltage of NF-sPLLA dried hydrogel scaffold is around 33.7 mV peak-to-peak, and superior to the negative control NF-sPDLLA dried hydrogel scaffolds (~5mV peak-to-peak).It is noteworthy that we utilized 10X PBS solution and NaOH to crosslink collagen hydrogel, making the liquid composite hydrogel become conductive due to the high concentration of salts.Because of the conductivity properties of the composite hydrogel in its liquid form, it is not feasible to directly measure the piezoelectric output of these hydrogels in their original wet form.Hence, we adopted a vacuum drying method to obtain dried forms of the composite hydrogels, which enabled accurate measurement of the piezoelectric output.This approach was demonstrated in our previous publication, where dried scaffolds were utilized to measure the output voltage of 3D piezoelectric scaffolds [1].We also updated this information in the Materials and Methods section.Response: We thank the reviewer for the comment.To clarify, in our study, we selected COL2A1, ACAN, SOX9, and GAG as markers to assess chondrogenesis in vitro, because they are known to be crucial for cartilage tissue, both at the gene and protein level.Thus, when evaluating the ability of a biomaterial to promote cartilage formation, it is important to observe an increase in all these genes and proteins.Indeed, our data indicates that the Piezo + US group significantly upregulated all COL2A1, ACAN, and SOX9 genes, and also led to an increase in GAG production compared to both the control group and other sham groups.On the other hand, the other groups, including Non-Piezo with and without US, as well as Piezo without US, exhibited only partial upregulation of individual genes (SOX9, ACAN, or COL2A1), but not all three genes, compared to the control group.These results are consistent with previous research, where the presence of fibers alone or solely introducing ultrasound (US) stimulation did not enhance chondrogenesis [1,2].Therefore, we believe that the Piezo + US combination provides the best conditions for promoting chondrogenesis.
Also, at the current stage, this work is a proof of concept, demonstrating the effectiveness of using piezoelectric hydrogel combined with US activation for cartilage regeneration.So, we focused on the outcome of the experimental group rather than investigating the effect of each factor (e.g., US, NF-sPLLA, NF-sPDLLA) or how each of these effects chondrogenesis.In order to provide a deep understanding of gene trends across different groups, further investigation is required and could be out of scope of this work.Response: Thank you for the reviewer's comment.As suggested, we performed experiments to measure voltage output of different concentrations of NF-sPLLA dried hydrogel sensors under ultrasound (US) activation.The discussion of these data was added to the main text of manuscript and the figure was updated to the Supplementary Figure 5.
In these experiments, output voltage of the sensors was measured at 40 KHz and 0.33 Watt/cm 2 which was the same condition with in vitro and in vivo studies.However, due to the high electromagnetic interference (EMI) noise at 40 KHz, we further validated the results by measuring the piezoelectric voltage output at 1 MHz, where we were able to control and reduce EMI.Supplementary Figure 5 depicts that 5mg/ml of NF-sPLLA in hydrogel generated a significantly higher output voltage compared to the 1 mg/ml and 10 mg/ml concentrations.Also, a similar trend of output voltages was observed under 40 KHz.This result indicates that low amounts of NF-sPLLA in the hydrogel produces minimal piezoelectric charges, therefore showing little to no effect on chondrogenesis.On the other hand, an excessive amount of NF-sPLLA within the same volume of hydrogel leads to a high density of fibers.This high fiber density could increase the fiber membrane mass, reducing the mechanical vibration and/or likely cause the charges generated by the fibers to cancel each other out, leading to a reduction in the overall piezoelectric output voltage under US activation.Therefore, 5mg/ml NF-sPLLA in hydrogel is an optimal condition that provides the highest voltage output under US stimulation.Comment #5: How the authors choose the parameters of ultrasound activation for in vivo treatment.I wonder that how much electrical output or stimulation dose is effective for chondrogenesis is still unknown.
Response: Thank you for your comment.We have provided the explanation in the manuscript in Supplementary Discussions.
For clarification, the parameters employed for in vivo experiments were maintained identical to those utilized for in vitro studies, consisting of a 40 KHz ultrasound (US), 0.33 Watt/cm 2 and exposure for a duration of 20 minutes.These parameters were chosen based on the following reasons: -First, although 1-3 MHz US frequencies are commonly utilized for US therapy, to penetrate through knee joint and activate the piezoelectric properties of NF-sPLLA hydrogel, a low frequency (e.g., 40 kHz) is more suitable [1].This is because a lower tissue absorption rate is observed at lower frequencies [2].Regardless of the frequency employed, it is crucial to ensure that the intensity remains below 0.5 Watt/cm 2 , as low-intensity US which is considered safe for human use [3][4][5][6].-Second, the in vitro data (Figure 2. a-f) clearly demonstrates that the chosen US parameters were efficient in activating electrical charge in the Piezo hydrogel.This efficiency is evidenced by the upregulation of gene expressions (COL2A, ACAN, and SOX9), as well as the increased formation of GAG and Collagen II protein in the Piezo + US group, when compared with the control/sham groups.-Third, we also verified that the same US parameters applied in our study effectively activated the piezoelectric charge within the knee joints, as illustrated in Supplementary Figure 7.b.This additional evidence further supports the rationale behind our chosen US parameters for in vivo experiments.
Regarding the electrical output or stimulation dose for chondrogenesis, these parameters vary across different studies [7,8].Currently, there is no clear value on the optimal or effective electrical cue dose for promoting cartilage healing.However, based on our in vitro study, we have found that our chosen US parameters and the charge generated from our piezoelectric hydrogel are safe to promote adiposederived stem cells (ADSCs) proliferation and effective to facilitate their differentiation into chondrocyte cells.Therefore, we have decided to use the same parameters for our in vivo study.
Nevertheless, this study is currently in the exploratory stage, aiming to establish the proof of concept for our work.Consequently, determining the threshold or optimal dosage for the electrical output falls beyond the scope of this present investigation and requires further investigations.
applied.However, there is currently a lack of clear guidelines or comprehensive studies identifying/evaluating safe threshold parameters for ES use in tissue regeneration, particularly in cartilage healing.
To establish safe parameters for piezoelectric stimulation, careful consideration of various factors is required.Firstly, for piezoelectricity activation methods, vibration intensity or mechanical pressure applied to piezoelectric material should fall within a range that mitigates the risk of cartilage damage.Secondly, the magnitude of voltage output generated by the piezoelectric stimulation must adhere to the safe range for ES.In this regard, for US intensity, we utilized low intensity (0.33 watt/cm 2 ) which is safe for human use [2][3][4][5].Furthermore, with the intensity of US employed in our study, the voltage output generated is very low and comparable to that observed in Barker's study, which utilized ES (ranging from 15 to 500 mV) for cartilage regeneration in a rabbit model [6].On top of that, our in vitro data indicates that the US intensity and the resulting output voltage applied to ADSCs are biocompatible (Supplementary Figure 3.c) and do not cause any harm to rabbits after a two-month treatment period (Supplementary Movie 4).Collectively, we believe the parameter for piezoelectric stimulation applied in this study is safe.
The manuscript was revised that included this information.

Reviewer #2:
Comments: The research paper proposed an injectable piezoelectric hydrogel which could be injected into the joints directly.Then, ultrasound was incorporated to activate the piezoelectric hydrogel, generating electrical stimulation for cartilage regeneration.The utilization of this injectable hydrogel presents a minimally invasive procedure for implantation, decreasing the potential risks triggered by conventional invasive surgery.The strategy used for implantation is novel and the electrical stimulation effects of cartilage regeneration enabled via piezoelectric hydrogel-ultrasound system is satisfactory.Therefore, the manuscript deserves attention for publication.Nevertheless, some concerns listed below need to be handled properly before publication.
Response: We sincerely thank the reviewer for reading our manuscript carefully and the highly positive remarks.Furthermore, we appreciate the reviewer's suggestions, which helped us improve our manuscript.Please find below our detailed responses to the reviewer.
Comment #1: The title of the manuscript can be re-considered so the main content can be reflected precisely while the novelty is highlighted.For instance, there is no experimental evidence in this manuscript to support the "biodegradable" and the other "medical applications" (except the cartilage regeneration), despite the injectable piezoelectric hydrogel and this implantation strategy certainly have these potentials.
Response: We thank the reviewer for their feedback.Upon careful reconsideration of the manuscript's tile, we changed to a new title "Injectable And Biodegradable Piezoelectric Hydrogel For Osteoarthritis Treatment" We have retained the key term "biodegradable" in the title, as our hydrogel consists of collagen I and PLLA, both widely recognized as biodegradable materials [1,2].Additionally, our previous research demonstrated that scaffolds fabricated from collagen I and PLLA fibers degrade over time [3].In the revised version of the manuscript, we have also performed the degradation study of the NF-sPLLA hydrogel taken at 37°C over a period of 9 weeks, along with an accelerated degradation condition (80 o C), as supporting evidence of its degradability.As seen in Supplementary Figure 2.g, the volume of the NF-sPLLA hydrogel scaffolds gradually decreased over time.After week 9, under accelerated conditions, the hydrogels degraded, broke down, and lost their original structures.Comment #2: Section "Piezoelectric hydrogel for chondrogenesis in vitro study": In this section, three different concentrations of NF-sPLLA in hydrogel, i.e., 1, 5, and 10 mg/ml, were given to explore the optimal concentration for subsequent in vitro and in vivo biomedical experiments.As demonstrated in the manuscript: "At a higher amount of NF-sPLLA, there were more ACAN and SOX9 generated (increasing from 1.5 to 10-fold, from 1 mg/ml to 10 mg/ml).However, for COL2A1 gene, 5 mg/ml of the NF-sPLLA generated more COL2A1 genes compared to other groups."The 10 mg/mL seems to be more effective compared to 5 mg/mL since the group of 10 mg/mL can induce ACAN and SOX9 genes to the optimal state while only the COL2A1 gene exhibits the optimal state when 5 mg/mL is employed.Nevertheless, as elucidated in the manuscript: "Since collagen II is the most abundant (~ 95%) and important protein in the hyaline cartilage matrix, we selected NF-sPLLA concentration of 5 mg/ml for all following experiments and in vivo studies.", the group of 5 mg/mL was selected for subsequent experiments according to the collagen II.How can this contradiction be explained?
Another relevant concern is that I fail to find the data with respect to collagen II in the section discussing the optimal concentration.
Response: We thank the reviewer for the comment and sorry for the confusion.For clarification, we selected 5mg/ml concentration for subsequent experiments because of the highest production of collagen II and the overall best piezoelectric performance of this scaffold.
Collagen II is the main component of cartilage, which constitutes up to 95% of the collagens in the cartilage.Studies have revealed that COL2A1 functions as an extracellular signaling molecule capable of significantly suppressing chondrocyte hypertrophy by promoting integrin β1−SMAD1 interaction [1-3], which avoids cartilage calcification.This is important because in the process of regenerating articular cartilage, it is not only necessary for cells to differentiate into chondrocytes but also for them to stably maintain the hyaline cartilage stage, which is distinct from the growth plate zone.Additionally, COL2A1 is considered as an important extracellular signaling molecule that can regulate chondrocyte proliferation and metabolism, similar to soluble molecule signals [4,5].Moreover, the collagen II network plays a vital role in retaining proteoglycans within the cartilage matrix and is the most essential protein in the hyaline cartilage matrix [6].
In addition, per reviewer 1 suggestion, we assessed the piezoelectric performance of the hydrogel at different concentrations under ultrasound (US) stimulation.In these experiments, NF-sPLLA dried hydrogel sensors at various concentrations output voltage were measured at 40 KHz and 0.33 Watt/cm 2 which was the same condition with in vitro and in vivo studies.However, due to the high electromagnetic interference noise at 40 KHz, we further validated the results by measuring the piezoelectric voltage output at 1 MHz.Supplementary Figure 5 depicts that 5mg/ml of NF-sPLLA in hydrogel generated a significantly higher output voltage compared to the other 1 mg/ml and 10 mg/ml under both 40 KHz and 1 MHz.Collectively, 5mg/ml of NF-sPLLA hydrogel which provided the best overall piezo performance (compared to higher concentration 10 mg/ml nanofiber hydrogel), and produced the most COL2A1 gene expression was selected for all subsequent experiments and in vivo studies.
We already edited the main text to add the information.Comment #3: Section "Piezoelectric hydrogel induces cartilage healing in rabbit osteochondral defect model": In the section of animal experiment, three different sensors of polyimide, PLLA, PZT were selected at the beginning to confirm whether the 40 kHz ultrasound could penetrate through tissue and reach the target site.Why don't choose the piezoelectric PLLA and non-piezoelectric PDLLA based hydrogels which have been used to carry out the following animal experiment?The result can also be the direct evidence that the ultrasound certainly activate the piezoelectric hydrogel to generate electrical signal.When it comes to the data obtained from the ultrasound activated polyimide, PLLA, PZT sensors, as illustrated in Figure S6.b, the difference of output voltage was observed between PLLA and PZT.Nevertheless, it's been reported that PLLA and PZT have a huge disparity on the capability of electromechanical conversion.To be specific, PLLA features a piezoelectric constant of ~10 pC/N (https://doi.org/10.1002/adma.201802084)while the commercial PZT have a piezoelectric constant of 450-650 pC/N (https://doi.org/10.1093/nsr/nwac101).Under the activation of the ultrasound with same intensity and frequency, a more remarkable difference should be obtained on the output voltage of PLLA and PZT.However, the difference of output voltage demonstrated in S6.b is not that large.Can this phenomenon be explained as well?
Response: We thank the reviewer for their comment.We would like to clarify that the purpose of this experiment is only to validate the penetration ability of 40 kHz US through various tissues, including skin, muscle, and ligament, and reach the targeted defect site.We selected lead zirconate titanate (PZT) as a positive control because PZT is a commonly used piezoelectric material for many medical applications.It is important to note that when using a 40 KHz US transducer to measure piezoelectric response of materials, the carried-out data may tangle with electromagnetic interference (EMI) noise.Therefore, we utilized non-piezoelectric material (e.g., polyimide) to generate a baseline which is only subjective to EMI but does not exhibit piezoelectric properties.As seen in Supplementary Figure 7.b the polyimide sensor also shows some signals at 40 kHz, but purely EMI noise and not piezoelectric signal.However, PLLA sensor (made of the aligned nanofiber film) which has comparable dielectric constant to polyimide (2.7 for PLLA and ~3 for polyimide), produced significantly higher output voltage due to their piezoelectricity properties.Regardless, Supplementary Figure 7.b demonstrates that the 40 kHz US can effectively penetrate different tissues and reach the intended defect site, successfully activating the piezoelectric response of the PZT, PLLA sensors.
We did not use NF-sPLLA and NF-sPDLLA-based hydrogel sensors to carry out the experiments for the following reasons.(1) We were using a 40 KHz US transducer, and there is considerable amount of EMI noise that would interfere with the measured signals which are small from the dried hydrogels.
(2) The fibers inside the sensors made from the dried NF-sPLLA or NF-sPDLLA based hydrogels (i.e., chopped nanofibers mixed inside the collagen and dried) were separated from each other and randomly oriented; therefore, these sensors have much less piezoelectricity (in the bulk material) compared to the sensors fabricated from the aligned PLLA nanofiber films (i.e. the non-chopped nanofiber film as it is after the electrospinning process).Besides, the sensors used in these experiments were significantly small in size (5x5mm) to ensure they fit inside the knee joint defect.This small size further reduced the output signals.Therefore, the dried NF-sPLLA and NF-sPDLLA-based hydrogel sensors are not ideal for confirming 40 KHz US penetration as they both would produce very small response signals due to the low piezoelectric effect, the EM noise, and the small device size.It should be noted that we already provided evidence of the piezoelectric properties of NF-sPLLA hydrogel and the non-piezoelectric properties of NF-sPDLLA in Figure 1.e and f (in the original submission).
In Supplementary Figure 7.b, the data with PZT, PLLA and polyimide already served our purpose of verifying that the selected US frequency and intensity can penetrate the rabbit's knee joint and reach the defect.Hence, we believe it is unnecessary to repeat the experiment with the dried NF-sPLLA and NF-sPDLLA based hydrogel sensors.
Regarding the small difference between PZT and PLLA sensors, we agree that PZT possesses greater piezoelectric constants than ones of PLLA.This often leads to an assumption that PZT always produces a much higher output than PLLA.However, this is only true when the materials are stimulated by impact forces at low frequencies.For ultrasound transmission, especially for responding to the US, the output performance of the piezoelectric materials depends strongly on their acoustic impedance, which defines how well the US can be transmitted between different mediums (in our case, between tissues and the testing sensor).Indeed, PZT acoustic impedance is very high (34.7 MRayl) compared to the averaged acoustic impedance of tissues (~1.5 MRayl), leading to a major amount of US scattered or reflected to surrounding tissues instead of stimulating the material.In fact, for practical applications, PZT-based ultrasound transducers require a matching layer and a backing layer to receive/respond to the US effectively.On the other hand, PLLA's acoustic impedance (~2.3 MRayl) is closer to one of the body tissues, allowing more US to activate the materials.Therefore, the signal from the PLLA sensors is slightly smaller than PZT sensors in our ultrasound measurement.
This explanation was included in the Supplementary Discussions.
Comment #4: Section "Conclusion/Outlook": At the end of this section, three different advantages of the research were concluded, as illustrated in the manuscript: "we have presented, for the first time, a novel piezoelectric hydrogel which can (1) be injected into the body via a minimally invasive process to preclude implantation surgery, (2) self-generate electrical cues to promote cartilage and other tissue healing under US activation, and (3) eventually, degrade into safe degradation byproducts to avoid invasive removal surgery and any harm to the body."However, the corresponding data supports over the concluded advantage (3) and part of (2) (i.e., "other tissue healing") are absent, hence, these parts can't be involved in the conclusion as the highlights.
Response: We thank the reviewer for their comment.We have edited our writing to make the precise conclusion as presented below: "we have presented, for the first time, a novel piezoelectric hydrogel which can (1) be injected into the body via a minimally invasive process to preclude implantation surgery, (2) self-generate electrical cues to promote cartilage and potentially heal other tissues under US activation, and (3) eventually, degrade into safe degradation byproducts to avoid invasive removal surgery and any harm to the body." We kept claim (3) as this is explained and data is provided in comment #1.
Comment #5: The whole manuscript should be carefully rechecked to remove format errors and typos.For example, as illustrated in Figure 1.d, the numbers on the left axis are covered.Furthermore, there is an inconsistency between the scale bars on Figure 1.b and Figure 1.g-i.
Response: We thank the reviewer for their comment.We have revised all the figures and carefully revised the manuscript to remove errors and typos.

Reviewer #3:
Comments: The goal of this study is to evaluate the benefits of an injectable, biodegradable piezoelectric hydrogel for cartilage repair.It is interesting, building up on previous work (ref.55).However, I have the following points that need critical attention: Response: We thank the reviewer for supportive feedback.In addition, we greatly appreciate your comments and questions that have helped us to significantly improve our manuscript.We have addressed all comments, as seen below.

In vitro comments:
Comment #1: The authors should have used rabbit BM-MSCs instead of ADSCs as those are the local regenerative cells that will repopulate the defects following gel injection.
Response: We thank the reviewer for the comment.We agree that BM-MSCs are the local regenerative cells that likely will repopulate the cartilage defects following gel injection.However, scientific evidence has demonstrated that both adipose-derived stem cells (ADSCs) and BM-MSCs possess equivalent potential for differentiation into various tissue lineages, including cartilage, bone, and skeletal muscle [1][2][3].Furthermore, ADSCs offer distinct advantages, including their availability, accessibility and the ease to be expanded for in vitro experiments [4].On top of that, we want to emphasize that ADSCs were chosen just as a stem cell model to validate our hypothesis that piezoelectric charge can induce stem cells (in general) into chondrocyte phenotype in vitro (in comparison with the other control groups of using non-piezoelectric stimulation).Therefore, as long as we use the same stem cell source and the same cell-culture condition for all in vitro groups, our experiment outcomes still serve our purpose to indicate the effect of piezoelectric stimulation on chondrogenesis.
We also revised our manuscript with this clear explanation provided here.Comment #2: I could not find the details on the US conditions employed.Also, do they match with those applied in vivo?How can they be standardized so they would match?
Response: We thank the reviewer for the comment.For clarification, this information was provided in the Material and Methods of the original manuscript.
To maintain consistency between the in vivo and in vitro experiments, we applied the same parameters that were used in vitro to in vivo.In details, for in vitro treatment, we utilized an ultrasonic bath (Branson 2800 CPX series) that generated US at a frequency of 40 KHz and an intensity of 0.33 Watt/cm².The cells were exposed to this US for a duration of 20 minutes.To replicate these conditions in the in vivo study, we developed a similar system consisting of a 40 kHz ultrasound generator (Steminc) equipped with two 40 kHz bolt clamped Langevin transducers (Steminc) connected in series.The same intensity of US and treatment time were applied in the in vivo experiments to make sure comparability with the in vitro setup.
Comment #3: No data are provided on live/dead cells upon (i) US treatment, (ii) hydrogel application, and (iii) both treatments.
Response: We thank the reviewer for the comment.This information was provided in Supplementary Figure 3.c in the original submission.
Our experimental data reveals that both the Piezo and Non-Piezo groups, with or without US treatment, exhibit biocompatibility comparable to the control group (cells inside collagen only) in both short-term (1-3 days) and long-term (14 days) assessments.Notably, the viability of ADSCs in the Piezo + US group showed a significant increase on day 9 and day 14 compared to the other control/sham groups.This result is consistent with literature indicating that piezoelectric charges/electrical stimulation (ES) had a positive influence on cell growth [1][2][3].Additionally, we also performed a hemolysis study, as shown in Supplementary Figure 3.a-b, which indicated these hydrogels are highly safe with a low hemolysis rate (less than 5%) to satisfy the requirements of the International Standards Organization.We also want to emphasize that in this work, we employed low-frequency and low-intensity US, both in vitro and in vivo study.The parameters of US being employed are safe and optimal knee joint

In vivo comments:
Comment #4: I could not find the details on the amount of hydrogel applied.Does it match with the condition used in vitro?How can it be standardized so both would match (especially in terms of cell numbers: cell numbers in vitro to BM-derived regenerative cells in vivo)?
Response: We thank the reviewer for the comment.In terms of the amount of hydrogel applied in vivo, we utilized approximately 30 µl of hydrogel, completely filling the defect, which had a diameter of 4 mm and a depth of 2 mm (considered as a critical-size osteochondral defect in the rabbit's knee).Regarding of standardizing in vitro and in vivo condition: -For cell density and cell number we would like to emphasize that: 1) in this work, we use piezoelectric hydrogel which is cell-free and will generate charges under ultrasound stimulation to promote body's own cells to migrate and heal the defects.Therefore, matching the cells used in vitro with the BM-MSCs in the in vivo condition is irrelevant.2) Cell density or cell numbers that was used in vitro testing had allowed us to optimize fiber concentrations, confirm the US parameters that yielded positive outcomes, and validate our hypothesis before animal experiments.-For ultrasound intensity and frequency: we applied the same parameters for both in vitro and the in vivo experiments, which were 0.33 watt/cm 2 and 40 KHz, with a treatment time of 20 minutes.
We updated the manuscript which included relevant information.
Comment #5: Figure 5a: a. it is hard to understand how the US treatment on itself allows for a better adhesion of the hydrogels to the defects (regardless of piezo/non-piezo or even control); b. the hydrogel is made of unnatural compounds (PLLA, PDLLA, type-I collagen and not hyaline type-II collagen) so it is again difficult to understand that repair occurs with so much matrix formation just upon US (US-piezo-safO-2 months) versus non-US-piezo-safO-2 months.c.Non-piezo without US is better over time than with US, please explain.Piezo without US is worse over time, please explain (that would not be good if US do not work or are not well tolerated in patients).
Response: We thank the reviewer for the comment.a.To clarify, the images provided in the manuscript depict H&E and Safran-O staining, which were used to visualize tissue and cellular structures within the defects after 1 or 2 months of treatments.These images do not visualize hydrogels.Furthermore, our purpose of utilizing US treatment was to remotely activate the piezoelectric charge from NF-sPPLA hydrogel, rather than promoting adhesion of the hydrogel with native tissues.Therefore, US treatment did not have any relation with adhesion of hydrogels and native tissues.Please note that the matrix formation observed in the control group (defect only) or controls + US is solely the result of the body's attempt to repair the injured cartilage by depositing fibrous scar tissue [1,2].
b.The use of synthetic compounds as biomaterials for cartilage regeneration has shown promising results in previous research, including alginate, Heparin, and PEG-based hydrogel [1].Therefore, it is normal to deploy these compounds as scaffolds in tissue engineering.In this study, these compounds (PLLA + type-I collagen) and US were used as the vehicle to deliver electrical cue.Our primary hypothesis is that electrical charges generated by the proposed hydrogel stimulate stem cells differentiation into the chondrocyte phenotype.As shown in Figure 3, we believe that the generated piezoelectric charges from the hydrogel also stimulate stem cells to generate TGF-β1 which is one of important growth factors in cartilage healing.Therefore, so much more hyaline cartilage matrix was formed and integrated well with the native tissues in the Piezo + US group (compared to the Piezo -US and other groups), which validated our hypothesis.This finding emphasizes the potential of the piezoelectric hydrogel as a promising approach for facilitating cartilage regeneration and repair.
c.We want to emphasize that the images presented in Figure 5.a only show the histological staining (H&E and Safranin-O) of one animal per group, serving as representative data.Since some images could lead to incorrect conclusions at the first glance, we decided to replace those images and revise Figure 5.a We would like to elaborate on the representative images of Non-Piezo without US after 1 and 2 months.Even though 2 months had more tissue filing in the defect compared with 1 month, the defect was filtrated with fibrosis scar tissue (hot pink arrow).Additionally, the fibrosis tissue was detached from native tissue which was similar for 1 month data (violet markers).For the Piezo group, at 1 month time point, we observed chondrocyte-like cells were formed inside the defect, however, the newly formed tissue was detached from the native tissue and the cells did not pack in any cartilage structure.Therefore, with time, the tissue will either collapse and fall out of defect due to the unstable structure or turn into bony tissue which was similar to 2 months data.Hence, Non-Piezo and Piezo samples without US at 1-or 2-month time point are almost the same in terms of cartilage regeneration.Along with the representative data, Figure 5.b (which was provided in the original submission) depicts quantitative histology score for all animals in the groups, evaluated by professional pathologists.Based on the histology scoring data, there were no significant differences in terms of cartilage regeneration between the control and sham groups at both the 1-month and 2-month time points.This suggests that neither US treatment nor the piezoelectric hydrogel alone exerted any notable positive or negative effects on the cartilage healing process in the animals.However, the combination of Piezo + US, which generated electrical stimulation, demonstrated a significant improvement in the healing process.

Figure 1 .
Figure 1.e, Output voltage waveform of sensors made of our dried NF-sPLLA hydrogel scaffold (Piezo sample) and NF-sPDLLA hydrogel (Non-piezo sample) in collagen under US activation.f, Peak-to-Peak output voltage of sensors made of our dried scaffold NF-sPLLA (Piezo sample) and NF-sPDLLA (Non-piezo sample) in collagen under US activation (n=4).

1
Liu, Y. et al.Exercise-induced piezoelectric stimulation for cartilage regeneration in rabbits.Science translational medicine 14, eabi7282 (2022).Comment #3: In Figure 2a-d, the expression of four gene shows different trends in the Non-Piezo, Piezo, Non-Piezo+US and Piezo+US groups.Please explain the possible reason in details.

Supplementary Figure 2
. g, Degradation study of NF-sPLLA hydrogel at 37 o C and accelerated condition 80 o C both in media and PBS with and without US treatment (scale bar: 1 cm).1.Okada, T., Hayashi, T. & Ikada, Y. Degradation of collagen suture in vitro and in vivo.Biomaterials 13, 448-454 (1992).2.Le, T. T. et al.Piezoelectric nanofiber membrane for reusable, stable, and highly functional face mask filter with long-term biodegradability.Advanced Functional Materials 32, 2113040 (2022).

Figure 5 |
Figure 5 | Piezoelectric hydrogel induces cartilage healing, evaluated by histology assessment and mechanical testing in vivo.a, H&E staining and Safranin O/fast green and collagen II staining to evaluate the articular cartilage regeneration for sham (defect only), non-piezo/piezo hydrogels with and without US activation (1-2 months).Black arrows indicate newly formed cartilage tissues.Yellow markers indicate the new cartilage tissue which was well-integrated with the native host tissue.Hot pink arrows indicate fibrillation filling, red arrows indicate bony tissue and violet markers indicate the detachment of newly formed tissue from the host (scale bars: 500μm).b, ICRS histological evaluation, (n=4 knees for each group).The score was an average point from three independent professionals and a blinded evaluation.The data are expressed as data points with Mean ± SEM *p < 0.05.